Interleukin 1 potentiates agonist-induced secretion of beta-endorphin in anterior pituitary cells

Interleukin 1 (IL-1) has been shown to potentiate the release of beta-endorphin induced by secretagogues, including corticotropin releasing factor (CRF) and phorbol ester (TPA), in the mouse AtT-20 pituitary tumor cell line (Fagarasan et al., PNAS, 1989, 86, 2070-2073). In cultured rat anterior pituitary cells, pretreatment with IL-1 caused only a small increase in beta-endorphin release but significantly potentiated CRF-and vasopressin-stimulated beta-endorphin secretion. Vasopressin stimulates the secretion of beta-endorphin in normal pituitary cells but not in AtT-20 cells. However, treatment of AtT-20 cells with IL-1 induced the expression of vasopressin-mediated beta-endorphin release; this effect of IL-1 was reduced after depletion of protein kinase C by prolonged treatment with TPA. The enhancement of CRF-stimulated beta-endorphin release by IL-1 was also reduced in AtT-20 cells after depletion of protein kinase C, and after treatment with staurosporine. These findings indicate that treatment with IL-1 amplifies receptor-mediated responses to the major physiological secretagogues in normal corticotrophs, and initiates a secretory response to vasopressin in AtT-20 cells.     

Phorbol Ester-Enhanced Noradrenaline Secretion Correlates with the Presence and Activity of Protein Kinase C-α in Human SH-SY5Y Neuroblastoma Cells

Abstract: The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes α, ε, and ζ on noradrenaline (NA) secretion from human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31-7549 inhibited carbachol-evoked NA release (IC₅₀ 0.6 µ M ) but not 100 m M K⁺-evoked release. In addition, Ro 31-7549 inhibited the enhancement of carbachol- and K⁺-evoked release after pretreatment with 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) for 8 min, with IC₅₀ values of 0.7 and 2.4 µ M , respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH-SY5Y cells to phorbol 12,13-dibutyrate (PDBu) or bryostatin-1 caused down-regulation of PKC-α and PKC-ε but not PKC-ζ. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to either PDBu or bryostatin-1, conditions that caused down-regulation of PKC-α, but not PKC-ε or ζ. The PKC inhibitor Gö-6976 (2 µ M ), which has been shown to inhibit selectively PKC-α and β in vitro, also inhibited the TPA enhancement of carbachol- and K⁺-evoked NA release by >50%. These data suggest that in SH-SY5Y cells, the ability of TPA to enhance carbachol- and K⁺-evoked NA secretion is due to activation of PKC-\ga.     

Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways

The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50 approximately 10(-8) M). Accumulation of alpha-melanocyte-stimulating hormone (alpha-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10(-8) M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10(-8) M, whereas the inactive phorbol ester 4 alpha-phorbol at 5 X 10(-8) M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on alpha-MSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of Protein Kinase C-α and Translocation of the Myristoylated Alanine-Rich C-Kinase Substrate Correlate with Phorbol Ester-Enhanced Noradrenaline Release from SH-SY5Y Human Neuroblastoma Cells

Abstract: The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the α-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-ε from cytosolic and membrane-associated to cytoskeleton- and membrane-associated TPA had no effect on the cytosolic location of PKC-ζ. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 µ M ). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 n M ) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-α (but not -ε or -ζ). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-α.     

Structural requirements of lyngbyatoxin A for activation and downregulation of protein kinase C.

Structure-activity studies of novel synthetic analogues of lyngbyatoxin A reveal that the lactam ring but not the 7-linalyl moiety of lyngbyatoxin A is essential for the in vitro stimulation of protein kinase C (PKC). (-)-Indolactam V (ILV), which contains no hydrophobic substituent at C-7, or analogues containing either a linalyl or n-hexyl group at C-7 were equally efficacious in stimulating HeLa cell PKC in vitro and in competing with phorbol 12,13-dibutyrate for binding to PKC in intact cells. The hydrophobicity of alkyl groups at C-7, however, influenced the potency of these compounds to bind to and activate PKC. In addition, these compounds exhibited differences in their ability to translocate PKC. Lyngbyatoxin A (0.1 microM) like TPA induced a rapid translocation of PKC from the cytosol to the membrane and subsequently led to a sustained decrease in both cytosolic and membrane PKC activity. In contrast, (-)-n-hexylILV (0.1 microM) and (-)-ILV (1 microM) produced a transient and attenuated decrease in cytosolic PKC activity. At concentrations that produced half-maximal PKC stimulation, (-)-ILV did not cause any downregulation of PKC whereas lyngbyatoxin A and (-)-n-hexylILV led to 60% and 40% PKC downregulation, respectively. Western blot analyses with monoclonal antibodies to PKC isoforms indicated that reduction in PKC activity by chronic exposure to TPA or lyngbyatoxin A analogues could be explained by downregulation of PKC alpha. Constitutive expression of PKC beta and PKC gamma isoforms was low in HeLa cells and was not affected significantly by TPA or lyngbyatoxin A analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Propranolol stimulates histone phosphorylation by a putative PK-C in partially purified homogenate of rat testicular interstitial cells. A possible mechanism for increased testosterone secretion by propranolol.

The beta-adrenoceptor blocker propranolol stimulated testosterone secretion by rat testicular interstitial cells (Leydig cell-enriched preparation) in vitro at concentrations ranging from 10(-5) M to 10(-4) M. Treatment of these cells with H7 (20 microM), an inhibitor of protein kinase C, reduced the stimulatory effect of L-propranolol on testosterone secretion by about 5-fold. At concentrations ranging from 31.25 microM to 1000 microM, L-propranolol reduced [3H]phorbol 12,13-dibutyrate binding (IC50 = 75 microM) to rat testicular interstitial cells. At similar concentrations, L-propranolol displaced the binding of [3H]phorbol 12,13-dibutyrate to the homogenate of these cells by only 5%. These findings suggest that the effect of L-propranolol on [3H]phorbol 12,13-dibutyrate binding could be indirect, possibly by increasing the concentration of a chemical mediator interacting with the regulatory domain of protein kinase C. At even lower concentrations (10(-9) M to 10(-7) M), propranolol added directly to the reaction mixture with protein kinase C partially purified from rat testicular interstitial cells increases the phosphorylation of histone. This phosphorylation was comparable to that obtained with (25 microg/ml) phosphatidylserine. The D- and L-stereoisomers of propranolol were equally active. A complete reversal of this propranolol effect on histone phosphorylation was achieved with (20 microM) H-7. In the absence of Ca2+, propranolol was not able to phosphorylate the histone. Taken together, these results suggest that protein kinase C could be the putative kinase involved in this reaction and that its activation by propranolol may be due to interaction of the drug with the regulatory domain of the enzyme at a site differing from the site of interaction with phorbol 12,13-dibutyrate. The ability of propranolol to activate the putative protein kinase C could be related to its stimulatory effect on testosterone secretion by Leydig cells.     

Activation of Protein Kinase C Augments Peptide Release from Rat Sensory Neurons

Abstract: To determine whether protein kinase C (PKC) mediates release of peptides from sensory neurons, we examined the effects of altering PKC activity on resting and evoked release of substance P (SP) and calcitonin gene-related peptide (CGRP). Exposing rat sensory neurons in culture to 10 or 50 n M phorbol 12,13-dibutyrate (PDBu) significantly increased SP and CGRP release at least 10-fold above resting levels, whereas the inactive 4α-PDBu analogue at 100 n M had no effect on release. Furthermore, 100 n M bradykinin increased peptide release approximately fivefold. Down-regulation of PKC significantly attenuated the release of peptides evoked by either PDBu or bradykinin. PDBu at 1 n M or 1-oleoyl-2-acetyl- sn -glycerol at 50 µ M did not alter resting release of peptides, but augmented potassium- and capsaicin-stimulated release of both SP and CGRP approximately twofold. This sensitizing action of PKC activators on peptide release was significantly reduced by PKC down-regulation or by pretreating cultures with 10 n M staurosporine. These results establish that activation of PKC is important in the regulation of peptide release from sensory neurons. The PKC-induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia.     

Human osteoblast-like cell proliferation induced by calcitonin-related peptides involves PKC activity

The calcitonin peptides [calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin] share many biological actions, including activity on bone cells. In the present study, CT (10(-11) to 10(-9) M) stimulated [(3)H]thymidine incorporation in primary cultures of human osteoblasts (hOB), as already demonstrated for CGRP and amylin. RT-PCR analysis showed that the calcitonin receptor and the calcitonin receptor-like receptor are both expressed in hOB. In these cells, CT (10(-10) M) and amylin (10(-9) M), in contrast to CGRP (10(-8) M), did not increase cAMP production. All three peptides stimulated protein kinase C (PKC) activity. To evaluate PKC involvement in hOB proliferation, cells were incubated with phorbol 12,13-dibutyrate, a stimulator of PKC activity; cell proliferation was increased in a dose-dependent manner (EC(50) = 3.4 x 10(-8) M). Staurosporine (10(-9) M), a PKC inhibitor, blocked phorbol 12,13-dibutyrate-induced PKC activity and cell proliferation. Inhibition of PKC by staurosporine also counteracted the stimulatory effect of CT, CGRP, and amylin on hOB proliferation. From these data, it is deduced that the activation of PKC is important for hOB proliferation and that it is involved in the anabolic effect of CT peptides on bone.     

Differing roles of protein kinase C on the signal transduction of tumour necrosis factor-alpha and -beta on PANC-1 cells: In vitro autoradiographic investigation

We investigated alterations in protein kinase C (PKC) activity of PANC-1 cells following treatment with tumour necrosis factor (TNF)-alpha or TNF-beta by an in vitro autoradiographic method. Binding studies performed on whole cells using [³H]phorbol-12,13-dibutyrate (PDBu) as a ligand revealed strong activation of PKC by TNFs within 30 min. The effect was similar to that seen after 30 min treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). After treatment for 24 h, TNF-beta caused a marked down-regulation of PKC similar to that seen after 24 h treatment with TPA; significant activation persisted, however, in the cells treated for 24 h with TNF-alpha. Our data suggest that PKC activation may play a more important role in the TNF-alpha signal transduction pathway than in that of TNF-beta.     

Involvement of functional protein kinase C in the mitogenic response to the H-ras oncogene product.

Microinjection of purified protein kinase C (PKC) into Swiss 3T3 fibroblasts pretreated with the phorbol ester phorbol-12,13-dibutyrate restores the mitogenic response of the cells to phorbol-12,13-dibutyrate (G. Pasti, J.C. Lacal, B.S. Warren, S.A. Aaronson, and P.M. Blumberg, Nature [London] 324:375-377, 1986). Our present studies demonstrate that the mitogenic activity of the H-ras oncogene in H-ras p21-microinjected quiescent cells is markedly reduced under conditions in which PKC is downregulated by chronic phorbol ester treatment. The ability to reconstitute the mitogenic response upon microinjection of both H-ras p21 and PKC implies involvement of functional PKC in the mitogenic activity of the H-ras oncogene product.     

Benzodiazepine suppression of corticotropin-releasing factor (CRF)-induced beta-endorphin release from rat neurointermediate pituitary.

Dopamine and gamma-aminobutyric acid (GABA) inhibit POMC peptide release from the pituitary intermediate lobe, via interaction with D2 or GABA-A/benzodiazepine receptors. Here, we examined the effects of an antianxiety triazolobenzodiazepine, adinazolam, on corticotropin-releasing factor (CRF)-stimulated POMC peptide secretion from the rat neurointermediate pituitary. Neurointermediate lobes (NILS) were incubated with CRF (10(-7) M), then adinazolam (10(-8) or (10(-9) M) was added, with CRF remaining in the medium. Aliquots were removed at 15-min intervals and frozen for radioimmunoassay of beta-endorphin. Adinazolam alone did not significantly affect secretion as compared to controls or CRF alone. Adinazolam incubated with CRF led to significant inhibition of beta-endorphin secretion, as compared to CRF alone. In addition, adinazolam was as effective as dopamine or the CRF antagonist, alpha-helical CRF, in preventing CRF-induced beta-endorphin release. Adinazolam appears to act directly on the pituitary to suppress hormone release induced by a stress-related hypothalamic peptide.     

Down-Regulation of Pinealocyte Protein Kinase C: Effect on α1-Adrenergic Potentiation of β-Adrenoceptor Stimulation of Cyclic AMP Accumulation and Induction of Serotonin N-Acetyltransferase Activity

Treatment of rat pinealocytes with 4 beta-phorbol 12,13-dibutyrate down-regulated protein kinase C (PKC) activity. Loss of activity was concentration-dependent (50% loss at 8 x 10(-7) M after 18 h of treatment) and time-dependent (50% loss after 2 h with 3 x 10(-6) M). Phenylephrine, an alpha 1-adrenergic agonist, and phorbol esters unable to activate PKC did not down-regulate the enzyme. alpha 1-Adrenergic amplification of beta-adrenergic stimulation of cyclic AMP accumulation, a response previously shown to be mediated by PKC activation, was reduced by only 50% in cells in which PKC activity was down-regulated by approximately 95%. These data suggest that there is not a simple proportional relationship between the degree of activation of pinealocyte PKC and the alpha 1-adrenergic amplification of beta-adrenergic cyclic AMP synthesis. In down-regulated cells, alpha 1-adrenergic amplification of beta-adrenergic induction of serotonin N-acetyltransferase activity, a key cyclic AMP-responsive enzyme involved in the nocturnal synthesis of the pineal hormone melatonin, was unchanged. Thus, even though alpha 1-adrenergic amplification of cyclic AMP synthesis is impaired, sufficient cyclic AMP is generated to allow a full induction of serotonin N-acetyltransferase activity. This finding raises the important question of whether the alpha 1-adrenergic amplification mechanism has a physiological role in regulating melatonin synthesis in vivo.     

Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells

The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitude and duration, and release of ACTH and beta- endorphin immunoactivity. Isoproterenol increased both action potential frequency and hormone secretion. Raising the external calcium concentration increased the frequency and amplitude of the action potentials and stimulated secretion of ACTH and beta-endorphin immunoactivity. Thus, stimulation of secretory activity in AtT-20 cells was closely correlated with increased electrical activity. However, a complete blockade of action potential activity had no effect on basal hormone secretion in these cells. These results suggest that the mechanisms underlying stimulated hormone secretion are different from those responsible for basal secretory activity. It is proposed that the increased influx of calcium due to the increased action potential frequency initiates the stimulated release of hormone from these cells.     

Protein kinase C inhibits Ca2+ accumulation in cardiac sarcoplasmic reticulum

It is now recognized that phorbol esters are negative inotropic agents in mammalian heart which presumably act via stimulation of Ca2(+)-activated phospholipid-dependent protein kinase (PKC). The goal in the present study was to identify the underlying cellular processes. Digitonin-permeabilized cultured neonatal rat ventricular myocytes were used to study biochemical and functional effects of phorbol esters on cardiac sarcoplasmic reticulum (SR). These cells contracted spontaneously at 3 microM Ca2+. Beating was inhibited by 10 microM ryanodine and was insensitive to 1 microM nifedipine. Thus, beating behavior results from the phasic oscillation of Ca2+ transport by SR in this preparation. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), decreased frequency by 30%, suggesting that Ca2+ transport by SR had been reduced. Whereas cAMP stimulated the rate of oxalate-supported 45Ca2+ uptake 2-fold, phorbol esters, TPA, and phorbol 12,13-dibutyrate inhibited this process by about 45%. The effects of phorbols were specific: (a) the alpha-analogues of TPA and phorbol 12,13-dibutyrate were inactive; and (b) the phorbol esters had no effect on Ca2+ transport in cells that had been depleted of PKC. TPA decreased oxalate-stimulated Ca2+ uptake over the entire range of Ca2+ concentrations, from 0.1 to 10 microM, by at least 70% without shifting the half-maximal effective Ca2+ concentration. Taken together these results indicate that the effects of phorbol ester on cardiac contraction are due to decreased Ca2+ transport by the SR and that these responses are mediated by PKC. These studies support the interpretation that the negative inotropic effects of phorbol esters are due, in part, to decreased SR function.     

Activation of protein kinase C differentially regulates corticotropin-releasing factor-stimulated peptide secretion and cyclic AMP formation of intermediate and anterior pituitary cells in culture

The present study was aimed at investigating the effect of protein kinase C (PKC) activation on CRF receptor function of proopiomelanocortin (POMC) cells in culture. Incubation of tissues with the phorbol ester PMA selectively potentiated corticotropin-releasing factor (CRF)-stimulated ACTH secretion and cyclic AMP formation of anterior pituitary (AP) cells, while, in sharp contrast, it failed to similarly affect intermediate pituitary (IP) cells and AtT-20 corticotrophs exposed to CRF. Unexpectedly, however, long-term treatment of cultures with PMA, which depletes cell stores of PKC, resulted in a similar dramatic attenuation of stimulated peptide release from both corticotrophs and melanotrophs, while being without significant effect on cyclic AMP production. Exposure of cells to PMA did not change either basal or CRF-enhanced levels of POMC mRNA. We conclude that activation of PKC fails to synergize with CRF-mediated signalling in IP and AtT-20 cells, although optimal CRF receptor expression requires the presence of a functional kinase C pathway, thus suggesting cross-talks between both messenger systems.     

Protein Kinase C-Mediated Down-Regulation of Voltage-Dependent Sodium Channels in Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 12- O -tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), decreased [³H]saxitoxin ([³H]STX) binding in a concentration (IC₅₀ = 19 n M )- and time ( t _1/2 = 4.5 h)-dependent manner. TPA (100 n M for 15 h) lowered the B _max of [³H]STX binding by 53% without altering the K _D value. Phorbol 12,13-dibutyrate (PDBu) also reduced [³H]STX binding, whereas 4α-TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H-7 (an inhibitor of PKC), but not H-89 (an inhibitor of cyclic AMP-dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine-induced ²²Na⁺ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine-induced ²²Na⁺ influx by brevetoxin. These results suggest that an activation of PKC down-regulates the density of Na channels without altering their pharmacological features; this down-regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.     

Evidence that the Peripheral-Type Benzodiazepine Receptor Ligand Ro 5–4864 Inhibits β-Endorphin Release from AtT-20 Cells by Blockade of Voltage-Dependent Calcium Channels

The demonstrations that Ro 5-4864, a ligand selective for the peripheral-type benzodiazepine (BZD) binding site, inhibited cellular differentiation and proliferation and that occupancy of the peripheral-type BZD binding site likely mediated the observed BZD effects on diverse endocrine tissues suggested that Ro 5-4864 disrupted a common cellular regulatory event. Using a well-characterized anterior pituitary-derived tumor cell line (AtT-20 cells), which synthesizes and secretes adrenocorticotropic hormone (ACTH), beta-lipotropin hormone (beta-LPH), and beta-endorphin (BE), we have investigated the molecular mechanism of action of Ro 5-4864's capacity to alter BE secretion. Ro 5-4864 inhibits basal and induced BE release from AtT-20 cells, through a cyclic AMP-independent mechanism. Ro 5-4864 completely blocked the corticotropin-releasing hormone and forskolin-induced release of BE without altering the concomitant production of cyclic AMP. The addition to AtT-20 cells of CGP 28392, a dihydropyridine that has been demonstrated in other systems to specifically activate voltage-dependent Ca2+ channels, resulted in a cyclic AMP-independent, dose-related increase in BE secretion. This CGP-induced BE release was blocked by increasing concentrations of Ro 5-4864. In contrast to the capacity of Ro 5-4864 to block CGP-induced BE release, Ro 5-4864 lacked the capacity to block enhanced BE secretion due to the calcium ionophore A23187, which increases intracellular Ca2+ levels independent of the voltage-dependent Ca2+ channels. Our findings suggest that Ro 5-4864 inhibits BE secretion from AtT-20 cells through a blockade of the voltage-dependent membrane Ca2+ channels and this mechanism of action may be responsible for Ro 5-4864's diverse effects observed on other cell types.     

Phorbol ester modulates serotonin-stimulated phosphoinositide breakdown in cultured vascular smooth muscle cells

Stimulation of cultured rabbit aortic vascular smooth muscle cells (VSMC) with serotonin (5HT) induced a rapid generation of inositol phosphates from receptor-mediated hydrolysis of inositol phospholipids. Pretreatment of these cells with 500ng/ml of pertussis toxin for 24h prior to addition of 5HT reduced 5HT-induced formation of inositol phosphates. Phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutyrate (PDBu), are known to activate protein kinase C (PKC), but their role on cultured VSMC stimulated by 5HT has not been defined. TPA exhibited a rapid inhibition of 5HT-stimulated phosphoinositide breakdown, although 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD), an inactive phorbol ester, did not inhibit it. These data suggest that a guanine nucleotide inhibitory (Gi) protein couples 5HT receptor to phospholipase C and TPA modulates 5HT-stimulated hydrolysis of inositol phospholipids in cultured VSMC through activation of PKC.     

Phorbol esters inhibit low density lipoprotein processing by cultured human fibroblasts

A 24 h pretreatment of MRC5 fibroblasts with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induced a marked decrease in low density lipoprotein (LDL) internalization and degradation; the maximal effect (about 55% decrease) was observed for 10(-7) M TPA. LDL binding was reduced about 35-40%. A significant decrease (about 25%) in LDL internalization was observed after a 2 h incubation of cells with the drug, but longer incubation times (4-6 h) led to a greater effect. Another tumor promoter, phorbol 12,13-dibutyrate decreased LDL internalization by about 35%, whereas the non-tumor promoting 4 alpha-phorbol 12,13-didecanoate had no effect. The protein kinase C inhibitor alpha-cobrotoxin partially antagonized the inhibitory effect of TPA on LDL internalization. The non-phorbol tumor promoter mezerein, another protein kinase C activator, decreased LDL uptake by about 50%. Finally, it was found that TPA had no significant effect on the affinity of the receptor for the LDL. These results suggest a role for protein kinase C in the LDL pathway in cultured human fibroblasts.